Abstract : Very Short Patch repair systems prevent mutations resulting from deamination of 5- methylcytosine t
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Abstract : Very Short Patch repair systems prevent mutations resulting from deamination of 5- methylcytosine to thymine. The Vsr endonuclease is the key enzyme of this system, providing sequence specificity. We identified two genes encoding Vsr endonucleases from Neisseria gonorrhoeae FA1090, V.NgoAI and V.NgoAII, based on DNA sequence similarity to genes encoding Vsr endonucleases from other bacteria. After expression of the gonococcal genes in Escherichia coli, the proteins were biochemically characterized. The coding sequence for V.NgoAI consists of 414 bp and would encode a protein of 138 amino acids (aa) with a calculated molecular mass of 16 kDa; the coding sequence for V.NgoAII is 423 bp and would encode a protein containing 141 aa with a calculated molecular mass of 16.7 kDa. V.NgoAI and V.NgoAII were purified to apparent homogeneity using metal affinity chromatography. The endonuclease activity and specificity of V.NgoAI and V.NgoAII were determined. V.NgoAI was found to be multispecific and recognizes T:G mismatches in every tested nucleotide context whereas V.NgoAII recognizes T:G mismatches in the following sequences: GTGG, CTGG, GTGC, ATGC or CTGC. Alanine mutagenesis of conserved residues showed that Asp50 and His68 of V.NgoAI and Asp51 and His69 of V.NgoAII are essential for hydrolytic activity. Glu25, His64 and Asp97 of V.NgoAII and Glu24, Asp63 and Asp97 of V.NgoAI are important but not crucial for activity V.NgoAI and V.NgoAII. However, Glu24 and Asp63 are also important for the specificity of V.NgoAI. On the basis of our results, concerning features of Vsr endonucleases encoded by N. gonorrhoeae FA1090, we postulate that at least two types of Vsr endonucleases can be distinguished. - Slides
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